The present invention relates generally to the field of molecular biology. More particularly, certain embodiments concern methods and compositions comprising DNA segments and proteins derived from bacterial species. More particularly, the invention provides fnibA nucleic acid and FnBPA amino acid compositions from Staphylococcus aureus. Also disclosed are peptide epitopes and protein sequences comprising site-specifically-modified or truncated fibronectin (Fn) binding site domains, and antibodies derived from immunization of animals with these peptide epitopes and binding site domains. Various methods for making and using these antibodies, peptides and DNA segments, peptides and nucleic acid segments encoding modified ligand binding site domains, and native and synthetic proteins are disclosed, such as, for example, the use of antibodies and/or DNA segments as diagnostic probes and templates for protein production, and the use of antibodies, proteins, fusion protein carriers, peptides and nucleic acid segments in various pharmacological and immunological applications.
1.2.1 MSCRAMMs
Bacterial adherence to host tissue involves specific microbial surface adhesins of which a subfamily termed MSCRAMMs specifically recognize extracellular matrix (ECM) components. Many pathogenic bacteria have been shown to specifically recognize and bind to various components of the extracellular matrix in an interaction which appears to represent a host tissue colonization mechanism. This adherence involves a group of bacterial proteins termed MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) (Patti et al., 1994; Patti and Hxc3x6xc3x6k, 1994).
Several Fn binding MSCRAMMs have been isolated and characterized from different Gram-positive bacteria. Genes encoding Fn binding MSCRAMMs from Staphylococcus aureus (Signxc3xa4s et al., 1989), Streptococcus pyogenes (Talay et al., 1994; Hansky et al., 1992) and Streptococcus dysgalactiae (Lindgren et al., 1993) have been cloned and sequenced. The deduced amino acid sequences revealed 60-100 kDa proteins with very similar structural organization. The N-terminal signal sequence is followed by a long stretch of unique sequence which in some cases is interrupted by two copies of an approximately 30 amino acid long segment. The ligand binding site is located just N-terminal of a proline-rich domain, which is believed to anchor the proteins in the cell wall. This domain is followed by the sequence LPXTGX (SEQ ID NO: 1) which is a cell wall targeting signal (Schneewind et al., 1995), a stretch of hydrophobic residues representing a trans-membrane unit and a short C-terminal cytoplasmic domain containing a cluster of positively charged residues. The primary Fn-binding sites on these MSCRAMMs consist of 30-42 amino acid long motifs repeated 3-4 times, and most of the repeated units contain a consensus sequence (Lindgren et al., 1993; McGavin et al., 1993). This domain is composed of a unit of 37-40 amino acids, repeated three or four times (FIG. 1).
Recombinant proteins corresponding to the repeat regions from the different Fn binding MSCRAMMs are all capable of inhibiting the binding of Fn to different Gram-positive bacteria, including S. aureus, S. dysgalactiae and S. pyogenes (Joh et al., 1994). Furthermore, studies using individual synthetic peptides revealed that a number of the repeat units retain Fn-binding activity, and interfere with binding of Fn by all of the Gram-positive species tested. These data suggest that the binding sites in Fn for the different MSCRAMMs are either overlapping or closely spaced on the matrix protein.
The repeat regions have been overexpressed as recombinant fusion proteins in Escherichia coli where the recombinant Fn binding domains (rFnBD) are linked to a stretch of histidine residues which are utilized for affinity purification of the rFnBD proteins. These proteins have been designated as rFnBD-D, rFnBD-A, rFnBD-B, and rFnBD-F, respectively FIG. 1. The rFnBDs were found to exhibit similar binding kinetics and dissociation constants; for example, the dissociation constants of the four recombinant proteins binding to porcine Fn was determined by biosensor analysis to be in the low nM range with the dominant dissociation rates varying between 1xc3x9710xe2x88x924 and 6xc3x9710xe2x88x924xc2x7sxe2x88x921. Additionally, the recombinant proteins have been shown to have cross-species specificity and inhibit binding of Fn to many different bacterial cells (Joh et al., 1994).
The repeated units of the Fn binding domains of the different MSCRAMMs are strikingly similar, and appear to contain a consensus sequence (McGavin et al., 1991; House-Pompeo et al., 1996). The repeat units have a high number of acidic residues, and there are conserved hydrophobic and acidic residues at certain positions. Overall there is a high degree of sequence similarity between repeated units in a specific MSCRAMM as well as between MSCRAMMs from different species. Synthetic peptides, analogous to the repeated units, also bind Fn, and by amino acid substitution in these peptides it has been determined that all conserved residues are not needed for Fn binding (McGavin et al., 1991).
Fn is a disulfide-linked dimeric glycoprotein that is found in a soluble form in body fluids and a fibrillar form in the extracellular matrix. The primary biological function of Fn appears to be related to its ability to serve as a substrate for the adhesion of animal cells. This adhesion is mediated by a family of dimeric receptors which recognize and bind to specific sites in the central part of Fn. The primary binding sites in Fn for MSCRAMMs from Gram-positive bacteria has been localized to the Fn NH2-terminal domain (N29) (Mosher and Proctor, 1980; Speziale et al., 1984). This domain is composed of five type I modules which are about 45 amino acids in length. The structure of N29 is a series of anti-parallel xcex2-sheets stabilized by several disulfide bonds interspersed at regular intervals in the sequences (Potts and Campbell, 1994; Venyaminov et al., 1983). The ability to bind Fn is located exclusively within the C-terminal 20 amino acids of each D-motif (Huff et al., 1994; McGavin et al., 1993; McGavin et al., 1991). These amino acids contain the sequence GG(X3,4)(I/V)DF, which is present in repeated motifs of other Fn-binding adhesins, and within the Fn-binding A2 motif of S. dysgalactiae FnBA, changes to either of the GG or IDF sequences resulted in loss of Fn-binding (McGavin et al. 1993).
The S. aureus Fn-binding MSCRAMM contains an additional ligand binding site in an approximately 30 amino acid long segment which has been designated Du that encompasses the consensus sequence and is located N-terminal of the repeat region. This segment can also interact with Fn and its N-terminal domain (designated N29) (Jxc3x6nsson, 1992).
S. aureus possesses two tandem fnb genes, encoding Fn-binding proteins FnBPA and FnBPB (Jxc3x6nsson et al., 1991; Signxc3xa4s et al., 1989), each of which possesses three consecutive 37-or 38 amino acid D-motifs, designated D1, D2, and D3. In tandem, these motifs comprise a high affinity Fn-binding domain, D1-3. Synthetic peptides representing each motif are also individually capable of low affinity Fn-binding, and can competitively inhibit Fn-binding to S. aureus (Huff et al., 1994; Signxc3xa4s et aL, 1989).
1.2.2 Attempts to Generate Antibodies That Block Fn Binding Have Failed
In all of the Fn-binding MSCRAMMs identified so far, the primary ligand binding sites have been located to domains composed of a 37-42 amino acid motif repeated 3-5 times (McGavin et al., 1993). Unfortunately, attempts to generate blocking antibodies employing both synthetic peptides and different forms of the D1-3 immunogen have been largely unsuccessful (Ciborowski et al., 1992; Rozalska et al., 1994; Speziale et al., 1996). Previous attempts to generate high affinity antibodies that could block S. aureus binding to Fn have had little success. For example, when rabbit polyclonal antibodies were generated to both a recombinant form of the S. aureus Fn-binding MSCRAMM rFnBD-D and to the Fn-binding synthetic peptide D2, although the titer and specificity of these antisera were good, and the IgG isolated from both immune sera recognized their respective antigens in a Western blot, the isolated IgG from the immune sera did not inhibit the binding of S. aureus to Fn.
Interestingly, antibodies purified from mice immunized with a xcex2-D-galactosidase Fn-binding MSCRAMM fusion protein did not block the binding of S. aureus to 125I-Fn (Ciborowski et al., 1992). However, antibodies raised against the xcex2-D-galactosidase Fn-binding MSCRAMM fusion protein that had been treated with formalin possessed moderate blocking activity. These findings suggest that modification of the antigen""s protein structure was a critical factor in determining its ability to generate blocking antibodies.
It is possible that the S. aureus Fn-binding MSCRAMM interacts with Fn via an xe2x80x9cinduced fitxe2x80x9d binding mechanism. Data supporting the concept of an xe2x80x9cinduced fitxe2x80x9d mechanism of Fn binding by Fn-binding MSCRAMMs has recently been obtained by both immunological (Speziale et al., 1996) and physical biochemical (House-Pompeo et al., 1996) techniques.
A monoclonal antibody 3A10, isolated from a mouse injected with the S. dysgalactiae Fn-binding MSCRAMM FnBA, was recently identified and characterized. The epitope for 3A10 was localized to a newly-identified Fn binding motif (designated Au) just upstream of the repeat domain of the primary ligand binding site on FnBA. The antibody 3A10 enhanced Fn binding to Au rather than inhibiting the binding. This effect was demonstrated in two different assay systems. First, 3A10 elevated the ability of the Au-containing proteins and synthetic peptides to compete with bacterial cells for binding to Fn. Secondly, 3A10 dramatically increased the binding of biotin-labeled forms of the Au-containing proteins to the ligand immobilized on a blotting membrane. Purified 3A10 IgG did not recognize the antigen by itself, and Fn was required for the immunological interaction between the antibody and the epitope. This induction effect of Fn was shown in both Western blot and ELISA analyses in which immobilized Au-containing molecules were probed with 3A10 with varying concentrations of Fn. These data and subsequent biophysical studies indicate that the ligand binding sites of FnBA has little or no secondary structure. However, when encountered by Fn, they appear to undergo a structural rearrangement resulting in expression of a ligand-induced binding site (LIBS). The data revealed that 3A10 specifically recognized a LIBS generated by complex formation between Fn and FnBA.
1.3 Deficiencies in the Prior Art
The emerging antibiotic resistance in many bacterial species including Staphylococcus aureus and various staphylococcal and streptococcal species is being seen as a potential threat to mankind (Begley, 1994). Particularly worrisome is the multidrug resistance of Staphylococcus aureus. Today, almost half of the staphylococcal strains causing nosocomial infections are resistant to all antibiotics except vancomycin and it appears to be only a question of time before vancomycin will become ineffective (Begley, 1994). It is in this scenario that one must consider new strategies in attempts to prevent and treat infection. A detailed knowledge of the molecular pathogenesis of infections caused by staphylococci and streptococci would provide an important base in the design of these strategies. MSCRAMMs mediating microbial adhesion to the host tissues are an attractive target in the development of new antimicrobial agents since it appears to represent a critical first step in the pathogenic process of most infections.
It is clear that while a variety of approaches to the treatment of bacterial diseases have experienced some success, the growing problems of antibiotic resistance, variability of antigens between species and in the same species through mutation of antigens, and the inefficient immune systems in young children, the elderly and other immunocompromised patients, all present difficulties that need to be overcome. Thus, there exists today an immediate need for an effective treatment for streptococcal and staphylococcal pathogens that can be used for a variety of infections in both man and animals.
Burnham and coworkers reported monoclonal antibodies (mAbs) were raised to the native D1-D4 epitope protein sequence (WO 94/18327), however the biological or possible inhibitory activity of these mAbs was not demonstrated.
Interestingly, antibodies purified from mice immunized with a xcex2-galactosidase Fn-binding MSCRAMM fusion protein did not block the binding of S. aureus to 125I-Fn (Ciborowski et aL, 1992). However, antibodies raised against the xcex2-galactosidase Fn-binding MSCRAMM fusion protein that had been treated with formalin possessed moderate blocking activity. These findings suggest that modification of the antigen""s protein structure was a factor in determining its ability to generate blocking antibodies. Formalin treatment is a general method of inactivating proteins by inducing Schiff bases in lysine residues. However, the reaction is reversible and the molecule can potentially regain functionality, and therefore negate the beneficial effects of such formalin treatment.
There are several possible reasons for these results, e.g., when an active Fn-binding MSCRAMM is injected into an animal, one may assume that the bacterial protein immediately complexes with Fn. In such a complex, the surface of the MSCRAMM representing the binding site will be occupied and not available for immunological recognition. If this argument is correct, it would represent a clever previously unknown strategy used by microbes to avoid host defense mechanisms.
The fact that it has not been possible to generate blocking antibodies to the native Fn binding proteins has precluded their use in active and passive immunization methods directed toward preventing bacterial infection. Therefore, the creation of site-specifically-modified epitopes in the Fn-binding MSCRAMMs which would lead to the creation of antigens that generate high-affinity blocking antibodies would represent a significant breakthrough in the field of infectious disease, and in particular, revolutionize the manner in which staphylococcal and streptococcal diseases are treated, thereby obviating the need for antibiotic therapies to treat bacterial infection. Likewise, the development of mAb groups which recognize multiple Fn-binding epitopes from a single FnBP would facilitate methods for inhibiting Fn binding to FnBPs using monoclonal antibodies. Methods of altering DNA sequences encoding peptide epitopes would provide a stable, reproducible means for incorporating changes in the peptide sequence, and therefore represent a superior advancement over current formalin denaturation of native proteins. The development of methods of inhibiting bacterial adhesion to host cells by inhibiting ECM component interactions with bacterial cell surface proteins would provide a marked improvement in the state of the art for treatment of such infections.
The present invention overcomes one or more of these and other drawbacks inherent in the prior art by providing novel compositions and methods for their use, for example in the treatment of bacterial infection and the prevention of bacterial adhesion using non-antibiotic strategies. The present invention provides, for the first time, antibodies that block the binding of fibronectin to fibronectin binding proteins. These antibodies are raised against peptides that, while based upon epitopes from the fibronectin binding domain of fibronectin binding proteins, do not bind to fibronectin. Thus, once introduced into an animal, these peptide epitopes do not form a complex with fibronectin. This allows antibodies to be made against the uncomplexed peptide epitope, which antibodies inhibit or block the binding of fibronectin to fibronectin binding proteins.
Disclosed are antibodies that inhibit or block the binding of a fibronectin binding protein to fibronectin, and peptide compositions that, upon immunization into a selected animal, produce such antibodies. Also disclosed are methods for the use of the novel peptide and antibody compositions in the treatment of microbial infections mediated by the inhibition of microbial binding to the host cell ECM component, Fn. Also disclosed are methods for active and passive immunization against microbial pathogens, such as streptococcal and staphylococcal pathogens, exemplified using peptide epitopes from the fibronectin binding domain of selected fibronectin binding proteins. Particular aspects of the invention relate to novel nucleic acid segments encoding these epitopes, and methods for the use of such nucleic acid segments in a variety of diagnostic and therapeutic regimens.
The invention first provides a composition comprising an antibody that binds to a fibronectin binding domain of a fibronectin binding protein and inhibits binding of the fibronectin binding protein to fibronectin. Thus in particular embodiments, these antibodies are referred to as blocking antibodies. In preferred aspects of the invention, the antibody binds to a peptide or epitope of the fibronectin binding domain that, when the peptide is not an integral part of the fibronectin binding domain, does not specifically bind to fibronectin. That is to say, in certain preferred aspects, the peptide itself, in isolation, does not bind to fibronectin.
In particular embodiments, the antibody binds to a fibronectin binding domain of a microbial fibronectin binding protein, preferably a streptococcal or a staphylococcal fibronectin binding protein. In other preferred aspects, the antibody binds to a fibronectin binding domain of a streptococcal Sfb, FnBA or FnBB or staphylococcal FnBPA or FnBPB fibronectin binding protein. In still other preferred aspects, the antibody binds to a fibronectin binding domain of a fibronectin binding protein expressed from a staphylococcal fnbA gene, particularly the fnbA gene of Staphylococcus aureus. 
Both polyclonal antibodies and monoclonal antibodies that inhibit the binding of fibronectin binding proteins to fibronectin are provided herein. Preferred are antibodies that bind to the same epitope as monoclonal antibody 9C3 or 11A5, including the monoclonal antibodies 9C3 or 11A5 themselves.
In certain embodiments, the antibody is linked to a detectable label, such as, but not limited to, a radioactive label, a fluorogenic label, a nuclear magnetic spin resonance label, biotin, avidin or an enzyme that generates a colored product upon contact with a chromogenic substrate, including, but not limited to, alkaline phosphatase, hydrogen peroxidase or glucose oxidase enzyme.
In certain aspects of the present invention, the antibody composition is dispersed in a pharmaceutically acceptable excipient or medium.
The invention further provides a composition comprising an isolated peptide of a fibronectin binding domain of a fibronectin binding protein, wherein the peptide does not bind to fibronectin. As used herein in regard to the instant isolated peptide compositions, the term xe2x80x9cof a fibronectin binding domain of a fibronectin binding proteinxe2x80x9d will be understood to mean that the isolated peptide is sufficiently similar to a portion of the wild-type sequence of the fibronectin binding domain of the fibronectin binding protein to allow the generation of an antibody that binds both to the isolated peptide and the fibronectin binding domain. Included within this description are isolated peptides wherein the sequence of the isolated peptide is or has been mutated, or is different enough to prevent binding of the isolated peptide to fibronectin.
Thus isolated peptide fragments from wild-type or naturally occurring variants and synthetic or recombinant peptides corresponding to wild-type, naturally occurring variants or introduced mutations that do not correspond to a naturally occurring fibronectin binding domain of a fibronectin binding protein are encompassed in the present invention. In preferred aspects, the isolated peptide comprises at least a first mutation as compared to the corresponding amino acid sequence from a wild type fibronectin binding domain. In yet other embodiments, the isolated peptide has been engineered to comprise the mutation. Included within the term xe2x80x9cengineeredxe2x80x9d are mutations introduced into the peptide, for example through peptide synthesis or site-directed mutagenesis.
The isolated peptides only need be of a sufficient length to allow for the generation of an antibody that binds both to the isolated peptide and the fibronectin binding domain, and blocks the binding of the fibronectin binding protein to fibronectin. In certain aspects, peptides comprising at least about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 22, about 24, about 25, about 30, about 35, about 40, about 45 or about 50 contiguous amino acids are preferred. In other preferred aspects of the invention, the isolated peptide comprises at least about 6 contiguous amino acids from the wild type sequence of the fibronectin binding domain. In more preferred embodiments, the isolated peptide comprises a contiguous amino acid sequence of at least about 8 amino acids from SEQ ID NO:60 or SEQ ID NO:61. In still more preferred aspects, the isolated peptide comprises the contiguous amino acid sequence of SEQ ID NO:60 or SEQ ID NO:61.
In aspects of the invention, the isolated peptide compositions are used to generate an immunological response in an animal. In these aspects, the compositions preferably further comprise an adjuvant. Furthermore, in these aspects of the invention, the isolated peptide composition is preferably dispersed in a pharmaceutically acceptable excipient.
In particular embodiments of the present invention, the isolated peptide is operatively linked to a selected amino acid sequence. Thus the invention provides compositions comprising a fusion protein comprising at least a first peptide of a fibronectin binding domain of fibronectin binding protein operatively linked to a selected amino acid sequence, wherein the first peptide does not specifically bind to fibronectin. In preferred aspects, the first peptide is operatively linked to a selected carrier molecule or amino acid sequence, including, but not limited to, keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).
Further provided is a composition comprising an isolated nucleic acid segment that encodes a peptide of a fibronectin binding domain of a fibronectin binding protein, wherein the peptide does not specifically bind to fibronectin.
The invention also provides a pharmaceutical composition comprising, in a pharmaceutically acceptable excipient, an effective amount of an antibody that binds to a fibronectin binding domain of a fibronectin binding protein and inhibits binding of the fibronectin binding protein to fibronectin, an isolated peptide of a fibronectin binding domain of a fibronectin binding protein, wherein the peptide does not specifically bind to fibronectin, or an isolated nucleic acid segment that encodes a peptide of a fibronectin binding domain of a fibronectin binding protein, wherein the peptide does not specifically bind to fibronectin.
The compositions of the present invention are contemplated for use in the preparation of an immunizing formulation. Thus, the invention provides for the use of the instant compositions in the preparation of an immunizing formulation.
The present invention also contemplates the use of the compositions provided herein in the preparation of a medicament for the prevention or treatment of an infection in an animal, or in the preparation of a formulation that inhibits the binding of microorganisms to fibronectin. Thus the invention also provides for the use of the instant compositions in the preparation of a medicament for preventing or treating an infection or in the preparation of a formulation that inhibits the binding of microorganisms to fibronectin.
The present invention further provides a method of identifying a peptide of a fibronectin binding domain of a fibronectin binding protein that does bind to fibronectin, comprising contacting a candidate peptide with fibronectin under conditions effective to allow binding of fibronectin to the fibronectin binding domain of a fibronectin binding protein, and identifying a positive candidate peptide that does not bind to fibronectin.
Also provided is a method of generating an antibody that binds to a fibronectin binding domain of a fibronectin binding protein and inhibits binding of the fibronectin binding protein to fibronectin, comprising administering to an animal a pharmaceutical composition comprising an immunologically effective amount of an isolated peptide of a fibronectin binding domain of a fibronectin binding protein, wherein the peptide does not specifically bind to fibronectin.
In certain aspects of the invention, the pharmaceutical composition is prepared by contacting a candidate peptide with fibronectin under effective binding conditions, and identifying a positive candidate peptide that does not bind to fibronectin and dispersing the positive candidate peptide in a pharmaceutically acceptable diluent. In preferred aspects, a plurality of candidate peptides are contacted with fibronectin under effective binding conditions, and one or more positive candidate peptide(s) that do not bind to fibronectin are identified. The plurality of candidate peptides can be generated by mutagenizing a plurality of peptides, exemplified by, but not limited to, replacing each residue of a selected peptide with proline residues, or in other embodiments by fractionating or fragmenting a native or naturally occurring fibronectin binding domain of a fibronectin binding protein, for example by digesting the fibronectin binding domain with a selected protease. The positive candidate peptides thus identified can be, in certain aspects of the invention, linked to a carrier protein and/or admixed with an adjuvant, or dispersed in a pharmaceutically acceptable medium or excipient.
In yet other aspects, the pharmaceutical composition comprises an immunologically effective amount of a peptide having the amino acid sequence of SEQ ID NO:60 or SEQ ID NO:61. In certain preferred embodiments, the pharmaceutical composition is designed for administration to an animal that has, is suspected of having, or is at risk of developing a microbial infection.
The invention additionally provides a method for detecting a fibronectin binding protein in a sample, comprising contacting a sample suspected of containing a fibronectin binding protein with an antibody that binds to a fibronectin binding domain of a fibronectin binding protein and inhibits binding of the fibronectin binding protein to fibronectin, under conditions effective to allow the formation of immune complexes, and detecting the immune complexes so formed.
In particular embodiments of the invention, the fibronectin binding protein is expressed by a microorganism, exemplified by, but not limited to, a streptococcus or a staphylococcus, and the sample is suspected of containing the microorganism. In certain preferred aspects, the fibronectin binding protein is expressed by Staphylococcus aureus. As the fibronectin binding proteins expressed by microorganisms localizes to the cell surface, in preferred aspects the fibronectin binding proteins are detected on or at the surface of the microbial cells.
Also provided are kits comprising, in suitable container means, an antibody that binds to a fibronectin binding domain of a fibronectin binding protein and inhibits binding of the fibronectin binding protein to fibronectin, an isolated peptide of a fibronectin binding domain of a fibronectin binding protein, wherein the peptide does not specifically bind to fibronectin, or an isolated nucleic acid segment that encodes a peptide of a fibronectin binding domain of a fibronectin binding protein, wherein the peptide does not specifically bind to fibronectin.
In certain embodiments of the present invention, the kits comprise, in suitable container means, a first antibody that binds to a fibronectin binding domain of a fibronectin binding protein and inhibits binding of the fibronectin binding protein to fibronectin. Thus in certain aspects, the invention provides immunodetection kits. In particular aspects, the immunodetection kits further comprise an immunodetection reagent, such as a detectable label that is linked to the first antibody. In other preferred aspects, the immunodetection kits further comprise a second antibody that binds to the first antibody.
Also provided are therapeutic kits, which comprise, in suitable container means, a therapeutically effective amount of the antibody, the isolated peptide or the isolated nucleic acid segment is comprised in a pharmaceutically acceptable formulation. In preferred embodiments of the instant therapeutic kits, the antibody inhibits the binding of streptococci or staphylococci to fibronectin. In yet other preferred aspects, the pharmaceutically-acceptable formulation is suitable for topical, parenteral or oral administration.
The invention further provides a method of preventing or treating a microbial infection in an animal, comprising administering to the animal a therapeutically effective amount of a pharmaceutical composition comprising, an antibody that binds to a fibronectin binding domain of a fibronectin binding protein and inhibits binding of the fibronectin binding protein to fibronectin, an isolated peptide of a fibronectin binding domain of a fibronectin binding protein, wherein the peptide does not specifically bind to fibronectin, or an isolated nucleic acid segment that encodes a peptide of a fibronectin binding domain of a fibronectin binding protein, wherein the peptide does not specifically bind to fibronectin, wherein the pharmaceutical composition prevents or inhibits microbial infection in the animal. As used herein, the term xe2x80x9ctherapeutically effective amountxe2x80x9d will be understood as an amount effective to inhibit the binding of the fibronectin binding protein of the microbe to the fibronectin of the animal. In preferred aspects of the present invention, the method prevents, inhibits or treats streptococcal or staphylococcal infection or colonization in the animal or human subject.